The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

Abstract

Proteins L7 and L12 from 50S ribosomal subunits of Escherichia coli are required for peptidechain termination. This termination process is inhibited by thiostrepton. Since both thiostrepton-treated ribosomes and those depleted of L7 and L12 have a markedly reduced ability to form release factor UA[3H]A ribosome complexes, the binding of release factors to the ribosome appears to be the primary site of inhibition.The ribosomal proteins L7 and L12 of Escherichia coli have been shown to be involved in the elongation factor Tu (EF-Tu)-dependent binding of aminoacyl-tRNA to ribosomes (1, 2) and in the elongation factor G (EF-G)-dependent hydrolysis of GTP (3-5). These same reactions are also inhibited by the antibiotic thiostrepton (6)(7)(8)(9)(10)(11)(12). In addition, as initially suggested by Hamel et al. (1), L7 and L12 are also required for the enzymatic binding of fMet-tRNA to ribosomest (13). Since these reactions all involve hydrolysis of the -y-phosphate of GTP and since L7 and L12 do not affect ribosomal peptidyl transferase activity (1), it appears that L7 and L12 might be required for reactions involving GTP hydrolysis dependent on a soluble factor and the 50S ribosomal subunit. The hydrolysis of GTP is required for peptide-chain termination in vitro, as studied with reticulocyte release factor (RF) and ribosomes (14). Although no such requirement has been demonstrated for peptide-chain termination in E. coli in vitro, a protein factor, RF-3, and guanine nucleotides are known to influence the ribosomal binding of E. coli termination factors RF-1 and RF-2 (15). Recently the binding of EF-G to E. coli ribosomes, which requires L7 and L12, was shown to inhibit the interaction of RF-1 and RF-2 with ribosomes (16). For these reasons the effects of the ribosomal proteins L7 and L12 and the antibiotic thiostrepton were studied with respect to their influence on peptide-chain termination. The present report provides evidence that L7 and L12 are required for peptidechain termination in E. coli and that this process is inhibited by thiostrepton. MATERIALS AND METHODSMaterials. Ammonium chloride-washed ribosomes were isolated from E. coli B as described (17), and ribosomal subunits were prepared from these ribosomes by the procedure of Pestka and Nirenberg (18). The ribosomal proteins L7 and L12 were extracted from the 50S ribosomal subunits with an Abbreviations: L7 and L12, 50S ribosomal proteins according to the nomenclature of Kaltschmidt and Wittmann (38); release factors, RF-1, RF-2, and RF-3; elongation factor Tu, EF-Tu; elongation factor G, EF-G.

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

“…Studies during the 1970s first demonstrated the requirement of L7/L12 for the functions of a wide variety of essential translation factors, including G and non-G proteins, in protein synthesis in vitro (7,8,27,28). Subsequent studies isolated mutants of L7/L12, which altered the fidelity and speed of protein synthesis in vivo (29).…”

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

“…The L12 stalk entails ribosomal protein L10 (or its archaeal and eukaryotic orthologue P0 in the P-stalk) and multiple copies of protein L12 (P1/P2 in eukaryotes 1 ). The L12 stalk is required for translation, because it recruits to the ribosome the auxiliary translation factors, in particular translational GTPases, such as initiation factor 2, elongation factors Tu and G, and release factor 3 or their eukaryotic homologues [2][3][4][5][6][7][8] , and accelerates GTPase activity of some of them 3,[9][10][11] . The mitochondrial homologue of L12, MRPL12, not only has an important role in translation but also regulates the transcription in mitochondria 12 .…”

The requirement for the Escherichia coli ribosomal proteins L7 and L12 in release factor‐dependent peptide chain termination

“…A codondependent in vitro termination activity independent of the ribosomal proteins L7/L12 of <40% of that observed with complete ribosomes was observed in some experiments with ribosome cores prepared as in [l] in contrast to [5]. We have eliminated the trivial possibility that this finding is a result of the core preparations retaining significant amounts of L7/L12.…”

“…Ribosomal subunits were isolated from sucrose gradients or from heat-shocked bacteria [ 121. Ribosomal core particles derived from the 70 S or 50 S were prepared by the method in [l] using the conditions of [5].…”

“…A stringent requirement for the proteins was suggested at least for EF-G and EF-Tu [l-4] and RF-1 and RF-2 [5,6] in studies using either selective removal of L7/L12 from the ribosome or antibodies against these proteins. More recently however, it has been shown that the elongation factors EF-Tu and EFG can interact with a functioning ribosome and carry out their reactions in the absence of L7/L12, although at a reduced rate [7,8].…”